Amido-phenyl-α-D-glucopyranoside derivatives

ABSTRACT

Synthesized succinyl and glutaryl glucosamines, p-(succinylamido)-phenyl-α-D-gluco- and mannopyranosides, p-(glutarylamido)-phenyl-α-D-gluco- and mannopyranosides and p-(isothiocyanotophenyl)-α-D-gluco- and mannopyranosides are reacted with insulin to form corresponding glycosylated insulins containing from 1 to 3 glycosyl groups per insulin molecule. The novel glycosylated insulins resist aggregation and show significant activity in depressing blood sugar levels.

This application is a division of application Ser. No. 442,362, filedNov. 17, 1982, now U.S. Pat. No. 4,444,683.

BACKGROUND OF THE INVENTION

This invention relates to the preparation of glycosylated insulins. Moreparticularly, this invention relates to the preparation of glycosylatedinsulins and to novel intermediates to be used in preparing glycosylatedinsulins.

Various systems have been proposed for the delivery of insulin to adiabetic patient that will be more responsive to the needs of thepatient.

The bioengineering approach is directed towards design of insulininfusion pumps. Hundreds of diabetics presently use externalbattery-operated pumps. The pump injects insulin continuously through aneedle attached to a catheter inserted into a vein or into subcutaneoustissue. The flow can be adjusted manually when a change occurs in theamount of insulin needed. The units are usually worn on a belt orstrapped to a leg.

Still in an experimental stage are pumps that deliver an amount ofinsulin precisely determined by a sensor that measures blood glucoselevels. Though successful progress has been made in this area, thesepumps are still too heavy to be portable. Another difficulty is that thesystem needs an apparatus for the continuous sampling of blood, ananalyzer to determine the blood glucose level rapidly and continuously,a computer to analyze the results and to determine the appropriateinsulin dose, and an infusion pump to deliver insulin intravenously in amanner approximating the delivery by the beta cells of the pancreas.Efforts are underway to reduce the size of the system and prolong itssensor's life. A "vest pocket" model, a system the size of a cigarettepack containing glucose sensor, power source, computer, insulinreservoir and pump, has been reported by Elliot in J. Am. Med. Assoc.,241, 223 (1979).

Another obstacle at present is the lack of an accurate implantableelectrode to sense the concentration of blood glucose. Again, athrough-the-skin connection to the patient's blood stream for longperiods presents risks of infection and clotting problems. Also, theoccurring aggregation of insulin in the artificial delivery systemsposes a considerable problem since the aggregated insulin willprecipitate or crystallize out of solution, thereby reducing thebioavailability of the insulin in an insulin reservoir. In addition, theaggregated insulin can become lodged in the delivery needle and preventthe flow of insulin from the delivery system to the diabetic.

OBJECTS AND SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to prepare asemisynthetic insulin which will not aggregate as rapidly as nativeinsulin and, therefore, have a longer storage life.

It is also an object of the present invention to prepare novelintermediate compounds to be used in the preparation of non-aggregatingsemisynthetic insulins.

A still further object of the present invention is to prepareglycosylated insulins which possess significant biological activity indepressing blood sugar levels.

These and other objects may be accomplished by means of novelglycosylated insulins having one of the general formulae: ##STR1## wherem is an integer of 1 to 3, X and Z are different and are selected fromthe group consisting of --H and --OH and --Q-- is a dicarboxylic acidspacer group having the formula: ##STR2## where n is an integer of from2 to 6 and is preferably 2 or 3.

DETAILED DESCRIPTION OF THE INVENTION

It is known that insulin can be combined with maltose as taught byBrownlee et al, in Science, 206, 223 (1979). However, this derivative ofa disaccharide and insulin has been found not to possess any significantbioactivity in depressing blood sugar levels.

In the present invention, the intermediates prepared for coupling withinsulin all consist of a glucose or mannose monosaccharide coupled to aspacer group. The spacer groups are derived from dicarboxylic acids,acid anhydrides or phenyl amines or a combinations thereof. Theintermediates have the following general formula: ##STR3## wherein

Y is a member selected from the group consisting of H, ##STR4##

X is a member selected from the group consisting of --H, --OH or##STR5## and

Z is a member of the group consisting of --H or --OH,

with the proviso that when Y is --H, Z must also be --H and X must be##STR6## when X is --OH, Z must be --H and Y must be ##STR7## and

when Z is --OH, X must be --H and Y must be ##STR8##

and where n is an integer of 2 to 6.

Preferably n is an integer of 2 or 3 and the ##STR9## portion of thespacer is derived from succinic or glutaric anhydride.

The intermediates described by the above formula may be broken down intotwo subgroups.

The first subgroup is the glucosamine derivatives wherein Z is --H, Y is--H and X is ##STR10## wherein n is an integer of 2 to 6

The second subgroup is the N-succinyl orN-glutarylamido-phenyl-α-D-gluco- and mannopyranosides and thep-isothiocyanotophenyl-α-D-gluco- and mannopyranosides wherein X and Zare different and are selected from the group consisting of --H and --OHand Y is a member selected from the group consisting of ##STR11## and nis an integer of 2 to 6.

The starting materials for the preparation of the sugar plus spacerglycocylated intermediates are glucosamine and p-nitrophenyl-α-D-gluco-and mannopyranosides and are commercially available.

The glucosamine may be reacted directly with an acid anhydride. Sincethe preferred spacers are succinyl and glutaryl moieties, the remainderof the discussion will be directed toward these derivatives. However, byappropriate synthesis, the corresponding derivatives from adipic,pimelic and suberic acids may also be utilized.

The p-nitrophenyl-α-D-gluco- and mannopyranosides are first treated toreduce the nitro group to an amino group. They may then be reacted withsuccinic and glutaric anhydrides to produce the correspondingN-succinyl- and N-glutaryl derivatives.

The p-aminophenyl-α-D-gluco- and mannopyranosides may also be reactedwith thiophosgene to form the correspondingp-isothiocyanotophenyl-α-D-gluco- and mannopyranosides. The synthesis ofthese products are detailed in the examples which follow.

The glycosylated intermediates which follow are representative of thenovel pyranosides which may be used to couple with insulin. ##STR12##

The structure of the insulin molecule is well known. It consists of twopolypeptide chains A and B linked together by disulfide bonds ofcystine. The N terminal group of the A fraction is glycine (Gly A-1) andthe N-terminal group of the B fraction is phenylalanine (Phe B-1). BothN-terminal positions contain reactive free α-amino groups. Adjacent theC-terminal group of the B fraction is lysine having a free ε-aminogroup. It is believed that these free amino groups contribute to theproblem of aggregation of insulin molecules with their eventualprecipitation.

By blocking these groups with the above glycosylated intermediates, itwas believed that the bioactivity of the insulin would not be greatlyaffected and that aggregation could be significantly inhibited orprevented. In addition, it is believed that glycosylated insulins mayhave other properties which may contribute to a chemical-sustainedrelease mechanism for delivery of insulin to a diabetic in directresponse to a change in blood sugar levels without the need for externalor implanted sensing devices.

The reaction of the intermediates shown above was carried out byconversion of the carboxylic acid at the end of the spacer to a mixedanhydride through reaction with an alkylchloroformate and reaction ofthe mixed anhydride with the native insulin. The mixed anhydride reactswith one or more of the A-1, B-1 or B-29 free amino groups on theinsulin to form a mono-, di- or triglycosylated insulin via an amidelinkage The degree of substitution will depend on the molar ratio ofintermediate to insulin and on reaction condition including pH.Generally, the molar ratio of intermediate to insulin will vary from 2to 10. For purposes of reaction, a pH range of about 8 to 9.5 ispreferable.

Because of the complexity of the reaction, one will seldom produce theglycosylated insulin as a mono-, di- or trisubstituted derivative.Rather, a mixture will be obtained as shown in the following examples.

For purposes of description, the glycosylated insulins may be dividedinto three categories.

The first category is those insulins prepared from glycosamines having a##STR13## spacer and possessing the general formula ##STR14## wherein nis an integer of 2 to 6, m is an integer of 1 to 3 and wherein theglycosyl group is attached to the insulin through one or more of theα-amino groups of the A-1 glycine, B-phenylalanine or ε-amino group ofthe B-29 lysine moieties of the insulin molecule.

Representative insulins are (glucosamidosuccinyl-)_(m) insulin and(glucosamidoglutaryl-)_(m) insulin.

A second category encompasses the gluco- and mannopyranosides coupledwith a ##STR15## spacer having the general formula ##STR16## wherein Xand Z are different and are selected from the group consisting of --Hand --OH, n is an integer of 2 to 6 and m is an integer of 1 to 3, andwherein each glycosyl group is attached to the insulin by an amidelinkage through one or more of the α-amino groups of the A-1 glycine,B-1 phenylalanine or ε-amino group of the B-29 lysine moieties of theinsulin molecule.

Representative compounds include[p-(α-D-gluco-pyranosyloxy)-phenyl-N-succinamyl] insulin;[p-(α-D-glucopyranosyloxy)phenyl-N-glutaramyl]_(m) insulin;[p-(α-D-mannopyranosyloxy)phenyl-N-succinamyl]_(m) insulin; and[p(α-D-mannopyranosyloxy)phenyl-N-glutaramyl]_(m) insulin.

The third category is inclusive of gluco- and mannopyranosides coupledwith a ##STR17## spacer having the general formula ##STR18## wherein Zand X are different and are selected from the group consisting of --Hand --OH, and m is an integer of 1 to 3 and wherein each glycosyl groupis attached to the insulin by a thioamide linkage through one or more ofthe α-amino groups of the A-1 glycine, B-1 phenylalanine or ε-aminogroup of the B-29 lysine moieties of the insulin molecule.

Representative compounds include[p-(α-D-glycopyranosyloxy)-phenyl-thiocarbamoyl-]_(m) insulin and[p-(α-D-mannopyranosyloxy)-phenyl-thiocarbamoyl-]_(m) insulin.

The glycosylated insulins prepared according to this invention may beadministered to a diabetic in any conventional manner, i.e.,subcutaneous, intramuscular or intraperitoneal injection. The dosage maybe the same in terms of IU (international units) as will free or nativeinsulin. Since dosages vary widely according to the needs of thepatient, no attempt will be made to try to define dosage ranges. Thatwill be left to the judgment of the patient's physician. Generally,dosages of 2 mg of insulin per day are required for a 60 kg. man.

The following examples show the preparation of the intermediatecompounds, the preparation of glycosylated insulins, their bioactivityand ability to inhibit or prevent aggregation.

EXAMPLE I Preparation of N-succinyl glucosamine

Glucosamine hydrochloride (0.05 m. 10.78 g) was dissolved in 15 mls ofdouble distilled water and 0.05 m triethylamine (6.95 ml). To this wasadded, with stirring, succinic anhydride (0.05 m, 5.705 g) in 37.5 ml ofacetone. The resulting mixture separated into two phases and sufficientwater was added to bring both phases into a single solution. Thesolution was held at room temperature for four (4) hours for thereaction to be completed after which it was placed in a vacuum chamberand evaporated until a viscous, yellowish concentrated solution wasobtained. The concentrate was measured and diluted with a triple amountof glacial acetic acid resulting in the formation of a white precipitateof N-succinyl glucosamine. The product was separated from the aceticacid solution by filtration and washed with ethanol and then petroleumether. The yield of the resulting product was 39%. The product had amelting point of 174°-175° C. and a molecular weight within 2.5% of thecalculated mole weight of 279.26. The structure and molecular weightwere confirmed by IR, NMR and MS/GC spectra.

EXAMPLE II Preparation of N-glutaryl glucosamine

The procedure of Example I was followed using glutaric anhydride. Theproduct yield was 41%. The melting point was 195°-196° C. The calculatedmole weight was 293.27. Structure was confirmed by IR and NMR spectra.

EXAMPLE III Preparation of p-(succinylamido)-phenyl-α-D-glucopyranoside

In a first step, p-nitrophenyl-α-D glucopyranoside (14 m mole, 4.214 g)in 350 ml of methanol was reduced by mixing with ammonium formate (56 mmole, 3.54 g) and palladium on carbon particles at 25° C. The system wasflushed for four (4) hours with nitrogen after which it was filtered andthe filtrate was evaporated at a reduced pressure. The crudep-aminophenyl-α-D-glucopyranoside was purified by recrystallization inan ethanol-water (50:1) mixture. The yield was 71%. Its melting pointwas 169°-170° C. Structure and molecular weight were confirmed by IR andMS/GC spectra. The observed molecular weight was within 2.7% of thecalculated mole weight of 271.27.

Following the procedure of Example I, p-aminophenyl-α-D-glucopyranosidewas reacted with succinic anhydride to producep-(succinylamido)-phenyl-α-D glucopyranoside in a yield of 53%. Themelting point of the product was 178°-180°. Structure and molecularweight was confirmed by IR, NMR and MS/GC spectra. The observedmolecular weight was within 2% of the calculated mole weight of 371.34.

EXAMPLE IV Preparation of p-(glutarylamido)-phenyl-α-D-glucopyranoside

The procedure of Example III was followed using glutaric anhydride inthe place of succinic anhydride. The p-(glutarylamido)-phenyl-α-Dglucopyranoside was produced in a yield of 63% and a melting point of167°-168° C. Structure was confirmed by IR spectra and the calculatedmole weight was 385.37.

EXAMPLE V Preparation of p-(succinylamido)-phenyl-α-D-mannopyranoside

First, p-nitrophenyl-α-D-mannopyranoside was reduced top-aminophenyl-α-D-mannopyranoside using the procedure outlined inExample III. The product yield was 91% and the product melted at150°-153° C. The structure was verified by IR spectra.

The p-aminophenyl-α-D-mannopyranoside thus produced was reacted withsuccinic anhydride in the manner described in Example III to producep-(succinylamido)-phenyl-α-D-mannopyranoside having a melting point of65°-66° C. in 67% yield. Structure and molecular weight were confirmedby IR, NMR and MS/GC spectra. The observed molecular weight was within2% of the calculated molecular weight of 371.34.

EXAMPLE VI Preparation of p-(glutarylamido)-phenyl-α-D-mannopyranoside

The procedure outlined in Example V was followed substituting glutaricanhydride for succinic anhydride. The resultingp-(glutarylamido)-phenyl-α-D-mannopyranoside melting at 134°-136° C. wasproduced in a yield of 75%. The calculated molecular weight was 385.37.Structure was confirmed by IR spectra.

EXAMPLE VII Preparation of p-isothiocyanotophenyl-α-D-glucopyranoside

To a solution of p-(aminophenyl)-α-D-glucopyranoside in 80% aqueousethanol was added a molar excess of thiophosgene (CSCl₂). The reactionwas carried out at room temperature and was complete in a manner ofminutes. A crystalline product was obtained. The calculated mole weightwas 313.3.

EXAMPLE VIII Preparation of p-isothiocyanotophenyl-α-D-mannopyranoside

The procedure of Example VII may be followed substitutingp-(aminophenyl)-α-D-mannopyranoside for the correspondingglucopyranoside to produce p-isothiocyanato-phenyl-α-D-mannopyranoside.

In confirming the synthesis of the above combinations of glucosamine orp-aminophenyl-α-D gluco- and mannopyranosides with succinic and glutaricanhydrides, the following tests were utilized. An infraredspectrophometer (Beckman Microlab 620 MX Computing InfraredSpectrophometer) was utilized to determine the reaction between theamino group and the anhydride by detecting the presence of an amidebond. Samples were prepared as 0.5% (w/w) KBr pellets. The presence ofthe amino group prior to reaction was detected by the N--H bendingvibration at 1650-1580 cm⁻¹. The p-aminophenyl derivatives prepared bythe reduction of the corresponding p-nitrophenyl derivatives did notshow N--O stretching bands at 1580 cm⁻¹ and 1330 cm⁻¹ indicating thatthe reduction reaction was complete. The formation of the amide bond wasshown by the presence of a C═O stretching band at 1660 cm⁻¹ and a N--Hbending mode at 1600 cm⁻¹. A normal dimeric carboxylic C═O stretchingband was also found at about 1725 cm⁻¹. These data confirm a distinctamide band indicating the completion of the coupling reaction betweenthe amino and dicarboxylic acid anhydride reactants.

The molecular weights were determined by MS/GC spectra using LKB 9000SMS/GC spectrophometer interfaced with a DEC PDP 11/34 computer. Thevolatility of the carbohydrate derivatives was enhanced by usingtrimethylsilyl derivatization of the hydroxyl and carboxylic acidgroups. In all instances, the observed molecular weight of thetrimethylsilyl derivatives was within 2.5±0.5 of the calculatedtheoretical values.

The presence of the ##STR19## moiety was confirmed by proton MNR spectrausing a JOEL JNM-FX 270 Fourier Transform NMR spectrophometer. Thesamples were dissolved in D₂ O and sodium2,2-dimethyl-2-silapentane-5-sulfonate (DSS) was used as an internalreference. For example, in the p-(succinylamido-α-D-glucopyranoside, theproton signals of the methylene groups in the succinyl moiety wasobserved at δ=2.71 as a triplet. The peak area was proportional to thenumber of protons representing the four methylene protons of thesuccinyl moiety.

The melting points were determined by the capillary melting pointmethod.

The yield of the above glucose and mannose derivatives with thedicarborylic acid anhydrides varied between about 39 and 91%. Thevariation in yield is thought to be due to the use of a limited solvent(ethanol-water mixture) for recrystallization. The yield should increasewith the selection of a proper solvent for the recrystallizationprocedure.

The recoupling action of the above described glycosylamidocarboxylicacid derivatives with insulin is carried out via a mixed anhydridemethod wherein the mixed anhydride is not isolated from the reactionmixture. The glycosylamidocarboxylic acid is converted to mixedanhydride by reaction with isobutylchloroformate and the resulting mixedanhydride is reacted with a free amino group from the insulin moleculeto form an amide linkage. The procedure is described in general byErlanger et al in J. Biol. Chem., 228,713 (1957) and Arekatsu et al inJ. Immunal., 97, 858 (1966).

There are three primary sites available on the insulin molecule forreaction with the glycosylamido-carboxylic acid derivatives and theinsulin may be coupled with one, two or three of these derivatives.These available sites include the α-amino groups of the glycine (GlyA-1), and phenylalanine (Phen B-1) and the ε-amino group of the lysine(Lys B-29) portions of the insulin molecule. The pKapp values of thesegroups are: 8.0 for Gly A-1, 6.7 for Phen B-1 and 11.2 for Lys B-29.

Because insulin becomes denatured at too high a pH and to maintain theε-amino group of the Lys B-29 moiety in a less reactive protonatedstate, the pH of the coupling reaction between theglycosyl-amido-carboxylic acids and insulin was chosen to be between 7.5and 10 and preferably at 9.5. Therefore, the α-amino groups of the GlyA-1 and Phe B-1 positions are thought to be the primary reaction sites.However, trisubstituted glycosylated insulin may also be produced by theabove method since a free ε-amino group from the Lys B-29 moiety couldbe formed by deprotonation through the use of the highly nucleophilictri-N-butylamine added to complex the HCl produced during the anhydrideformation by an isobutylchloroformate. Also, based on theHenderson-Hasselbach equation, at a pH of 9.5, about 2% of the ε-aminogroups of Lsy B-29 exist in equilibrium in the free or deprotonatedform. Therefore, a significant amount of trisubstituted glycosylatedinsulin may be prepared. However, because of the pH chosen, i.e., 9.5and the more reactive free amino groups of Gly A-1 and Phen B-1 at thepH, the glycosylated insulin will be primarily a mixture of di andtri-substituted derivatives. Some monosubstitution may also be present.

In the following examples, the unreacted insulin is removed from theglycosylated insulin by means of affinity chromatography using a columncontaining Sepharose beads bound with Con-A (Concanavalin-A).

It is known that Con-A has a binding affinity for saccharides.Therefore, the more glycosyl moieties coupled to the insulin, thegreater that glycosylated insulin will be bound to the Con-A in thechromatography column. One would then expect the unreacted insulin to beeluted through the column first followed by mono-, di- andtri-glycosylated insulins in that order.

This is generally true. However, some glycosylated derivatives may beeluted from the column along with unreacted insulin.

The following example is typical of the process of separation unreactedinsulin from glycosylated insulin by affinity chromatography with Con-A.

EXAMPLE IX Preparation of N-succinylglucosamine Coupled Insulin(Glucosamidosuccinyl Insulin)

Bovine insulin (87.77 μmoles 500 mg) was dissolved in 200 mls of anequal volume mixture of distilled water and dimethylformamide (DMF) andadjusted to a pH of 9.5 with 0.1N sodium hydroxide and was then cooledin an ice bath. N-succinylglycosamine, (800 μmoles) was dissolved in asolution of DMF containing 800 μmoles each of tri-N-butylamine andisobutylchloroformate and kept at 0° C. for 20 minutes. An additional1.6 m mole of tri-N-butylamine was added to this solution which was thenmixed, with stirring, to the insulin solution. The reaction mixture thusformed was pH adjusted to 9.5 with 0.1N sodium hydroxide and kept forone hour at 0° C. The mixture was then kept at room temperatureovernight and then dialyzed through a semipermeable membrane for twodays against distilled water to remove unreacted N-succinylglucosamine.The distilled water was maintained at 4° C. and was changed every fourhours.

The glycosylated insulin remaining inside the dialysis membrane waslyophilized and dissolved in the tris-buffer solution described below.The resulting solution was sterilized by filtration to remove anybacteria present.

The sterilized product was placed on a 2.5×60 cm column containing beadsof commercial Con A (Concanavalin-A) bound to Sepharose 4B (SigmaChemical Co., St. Louis, Mo.). The unreacted insulin was removed fromthe column using a 0.02 m tris-buffer eluent also containing 1 mm MnCl₂,1 mm CaCl₂ and 0.5 m NaCl. The eluent had a pH of 7.4 and was maintainedat 4° C. The flow rate was maintained at 72 ml/hr and 7.0 ml fractionswere collected and analyzed by UV spectra at A 276 nm for the presenceof insulin. A colorimetric determination for sugars at 480 nm using aphenol-sulfuric test also showed the presence of someN-succinylglucosamine coupled insulin.

After approximately 105 minutes as shown by FIG. 1, all of the unreactedinsulin (component 1) had been collected as monitored by the UV spectraat 276 nm. At that time, 0.1 m α-methyl-D-mannopyranoside was added tothe tris-buffer solution as an eluent and the flow rate was maintainedat 72 ml/hr. After approximately 200 minutes, all of component 2,consisting of N-succinylglycosamine, coupled insulin, had been collectedas also shown in FIG. 1.

The low intrinsic binding capacity of the glucosamine moiety to Con-Awas thought to be responsible for the mixed elution to free insulin andglycosylated insulin in component 1. Due to the low absorptivity of theglycosylated insulin in Component 2 at 480 nm, the degree ofsubstitution could not be determined.

The glycosylated insulin in component 2 was lyophilized fordetermination of its ability to depress blood sugar levels.

The corresponding N-glutarylglucosamine coupled insulin(glucosamidoglutaryl insulin) was prepared in a similar manner.

EXAMPLE X Preparation of p-(succinylamido)-phenyl-α-D-glucopyranosideCoupled Insulin [p-(α-D-glucopyranosyloxy)-phenyl-N-succinamyl insulin]

The procedure of Example IX was followed for reacting thep-(succinylamido)-phenyl-α-D-glucopyranoside from Example III withbovine insulin. The results are shown in FIG. 2. Component 1 in FIG. 2consisted of free insulin and some glycosylated insulin as verified bythe phenol-sulfuric acid method at 480 nm. Components 2 and 3 werecollected and tested by the phenol-sulfuric acid method for the presenceof the glycosyl radical as well as at 276 nm for insulin. Due to thelarge amount of eluent required to separate component 3, it can bepredicted that Component 3 contained more glycosyl radicals on theinsulin than Component 2. The area under the curves of Components 2 and3 was 58.9% and 41.1% respectively. Component 2 was primarily diglycosylsubstituted insulin and Component 3 was primarily the triglycosylsubstituted derivative. Therefore, 0.589×2+0.411×3=2.411 which would bethe average number of glycosyl derivatives on the insulin contained inComponents 2 and 3 combined. This degree of substitution was consistentwith the phenolsulfuric acid test which showed 2.3 glycosyl groups perinsulin molecule. The phenol-sulfuric acid test is detailed by Dubois etal, Analytical Chemistry, 28, 350 (1956).

After collection Components 2 and 3 were combined and dialyzed to removethe eluent, α-methyl-D-mannopyranoside, the purified product waslyophilized for biological testing.

Following the same procedure, the correspondingp-(glutarylamido)-phenyl-α-D-glucopyranoside coupled insulin[p-α-D-glucopyranosyloxy)-phenyl-N-glutaramyl insulin] was prepared.

EXAMPLE XI Preparation of p-(succinylamido)-phenyl-α-D-mannopyranosideCoupled Insulin [p-(α-D-mannopyranosyloxy)-phenyl-N-succinamyl insulin]

The procedure outlined in Example X was followed and the elution profileis shown in FIG. 3. Component 1 was unreacted free insulin since thephenol-sulfuric acid test was negative. The average degree of glycosylradicals attached to insulin for the combination of components 2 and 3was 2.5 according to the phenol-sulfuric acid test. The area under thecurves for components 2 and 3 was 34% and 66% respectively indicating anaverage degree of substitution of 2.66 which compares closely with theabove test results.

The purified lyophilized product was retained for testing for bloodsugar reduction.

The corresponding p-(glutarylamido)-phenyl-α-D-mannopyranoside coupledinsulin [p-(α-D-mannopyranosyloxy)-phenyl-N-glutaramyl insulin] wasprepared and purified by the above procedures.

EXAMPLE XII Preparation ofp-(α-D-glucopyranosyloxy)-phenyl-thiocarbamoyl Insulin

p-(Isothiocyanotophenyl)-α-D-glucopyranoside (355.08 μmoles) fromExample VII was dissolved in a solution of three parts pyridine and onepart water at 5° C. and the pH was adjusted to 8.0 with 0.1 N NaOH.Bovine insulin (177.54 μmoles, 1 gm) was prepared using a pyridine-watersolvent and combined with the glucopyranoside solution. The combinedsolutions were maintained at 5° C. at a pH of 8.0 for one hour and thenallowed to stand overnight at room temperature. The reaction productconsisting of p-(α-D-glucopyranosyloxy)-phenyl-thiocarbamoyl insulin wasthen dialyzed as in Example IX to remove unreactedp-(isothioxyanotophenyl)-α-D-glucopyranoside and the remaining productwas lyophilized, dissolved in tris-buffer and subjected to affinitychromatography on a Con-A Sephorase 4B column as in Example IX. The flowrate was 26 ml/hr at 4° C. and 5.0 ml fractions were collected. Theelution profile is shown in FIG. 4. Component 1 contained both freeinsulin and glycosylated insulin and Component 2 consisted ofp-(α-D-glucopyranosyloxy)-phenyl-thiocarbamoyl insulin having an averageof 1.5 glycosyl groups per insulin molecule.

The product from component 2 was dialyzed to remove theα-methyl-D-mannopyranoside eluent and was then lyophilized forbiological testing.

EXAMPLE XIII Aggregation Studies

One of the problems associated with free or native insulin is itstendency to aggregate and eventually crystallize out of solution,thereby reducing its bioavailability. With the glycosylated insulinsthis tendency is greatly reduced since portions of the active aminosites on the Gly A-1, Phe B-1 and Lys B-29 in insulin are blocked by thecoupling reaction of glycosyl groups.

Bulk aggregation studies with free insulin compared with glycosylatedinsulins were carried out by two methods. In a bulk aggregation study,various aqueous insulin and glycosylated insulin solutions containing0.1 mg/ml of insulin were stirred at 1555 rpm until aggregation wasvisually observed or up to two weeks. In a second test, solutionscontaining the same insulin concentration were deposited on polyurethane(Biomer) and microscopically observed for aggregation.

The results are as follows:

    ______________________________________                                        TIME REQUIRED FOR AGGREGATION                                                 Aggregation          Glycosylated Insulins                                    Test     Free Insulin                                                                              A        B       C                                       ______________________________________                                        Bulk     2-3 days    2 weeks  2 weeks 2 weeks                                 Polyurethane                                                                           1-2 days    2 weeks  2 weeks 8 days                                  ______________________________________                                         A = p(D-glucopyranosyloxy)-phenyl-N--succinamyl insulin                       B = p(D-mannopyranosyloxy)-phenyl-N--succinamyl insulin                       C = p(D-glucopyranosyloxy)-phenyl-thiocarbamoyl insulin                  

It is obvious from the above results that the glycosylated insulins aremuch more stable against aggregation than free insulin and will thushave a better storage life.

EXAMPLE XIV Bioactivity of Glycosylated Insulins

The bioactivity of the glycosated insulins described herein wasdetermined by a blood sugar depression test and compared to commercialinsulin preparations and controls. In this test, replicates of standardlaboratory rats were fasted for twenty hours. After measuring baselineblood sugar levels, a 1 mg/kg dose of either free or glycosylatedinsulin was injected via an intraperitoneal route. The blood sugar levelin each rat was measured colormetrically 20 minutes after the injection.The results are given in the following table:

    ______________________________________                                        BLOOD SUGAR DEPRESSION (BSD) TEST                                                                        Blood Sugar Con-                                                              centration after 20                                                    No.    min. IP. injection                                 Type of Insulin     Rats   (± S.E.M.) mg/dl                                ______________________________________                                        SIGMA INS (21F-0375)25.5IU/MG.                                                                    5      32.66 ± 1.10                                    LILLY INS (615-70N-80)100IU/MG.                                                                   5      31.87 ± 1.93                                    Glucosamidosuccinyl insulin                                                                       5      37.80 ± 1.13                                    (unpurified)                                                                  Glucosamidosuccinyl insulin                                                                       4      45.92 ± 3.30                                    (unpurified)                                                                  p-(α-D-glucopyranosyloxy)-phenyl-                                                           5      40.87 ± 1.32                                    N--succinamyl insulin                                                         p-(α-D-glucopyranosyloxy)-phenyl-                                                           5      40.40 ± 2.53                                    N--glutaramyl insulin                                                         p-(α-D-mannopyranosyloxy)-phenyl-                                                           5      44.80 ± 2.37                                    N--succinamyl insulin                                                         p-(α-D-mannopyranosyloxy)-phenyl-                                                           5      40.60 ± 0.80                                    N--glutaramyl insulin                                                         p-(α-D-glucopyranosyloxy)-phenyl-                                                           5      44.67 ± 2.21                                    Thiocarbamoyl insulin                                                         Control             *44    64.62 ± 0.61                                    ______________________________________                                         *Includes all rats used in test at baseline level.                       

It is evident from the above that the seven glycosylated insulinsprepared, as described herein, all possess significant biologicalactivity in depressing blood sugar levels.

What is claimed is:
 1. A compound of the formula ##STR20## wherein X andZ are different and are selected from the group consisting of --H and--OH and n is an integer of 2 to
 6. 2. A compound according to claim 1wherein Z is --H and X is --OH.
 3. A compound according to claim 2wherein n is
 2. 4. A compound according to claim 2 wherein n is
 3. 5. Acompound according to claim 1 wherein Z is --OH and X is --H.
 6. Acompound according to claim 5 wherein n is
 2. 7. A compound according toclaim 5 wherein n is 3.